A site where a man with far too much interest in beer gets to write about it.

Monday, 30 January 2017

The Brewing Science of Brettanomyces

The Brewing Science of Brettanomyces

The British Fungus

Brettanomyces is a genus of
non-Saccharomyces yeast of importance to the brewing industry.
It was named by NH Claussen, the Director of the Carlsberg
laboratory, in a paper published in the Journal of the Institute of
Brewing in 19041. He had identified from English stock
ales the organism responsible for “both the condition of these
beers and their flavour”. He named it Brettanomyces (British
fungus) due to its close connection to the British brewing industry.
Stock ales underwent a long maturation and for them to condition a
true secondary fermentation, by a secondary yeast, was necessary.
Using Hansen's pure yeast culture method for producing stock ales
obtained poor results as the pure cultures were free of
Brettanomyces. Demand for stock ales was however in terminal
decline by the time of Claussen's paper. Mild or running beers served
soon after brewing replaced them, and like the cask beers of today
they came into condition though the action of Saccharomyces on
residual or added priming sugars without the need for a secondary
fermentation by Brettanomyces.

Adding Brettanomyces to bring about a
secondary fermentation was however used in Courage Imperial Russian
Stout, and in Belgium, the method continues to be used in the
production of the Trappist beer Orval. It also plays an important
role in Belgian sour beer production, and recently interest in using
this yeast has grown, to the extent that some breweries now use it
for primary fermentation.

Orval Brett.

Current Classification

As with most micro-organisms the Brettanomyces
species have been reclassified as scientific understanding has
advanced. Brettanomyces had only been known to reproduce
asexually, but the discovery of ascopsore formation in Brettanomyces
by Van der Walt and Kerken2 led to its
reclassification of the genus as Dekkera. Species have also
been reclassified which adds to the difficulty of keeping up with
current nomenclature. At the present time there are four species3
in the genus, two of which have been seen to form spores:

The spore formers are Dekkera anomala (into
which the species claussenii has been merged) and Dekkera
bruxellensis (into which the species lambicus has been
merged) and the non-spore-formers are Brettanomyces custersianus
and Brettanomyces naardenensis. As it is likely that the genus
will be reclassified back to Brettanomcyes4,and
it is the most commonly used term by those that brew with these
organisms, I shall continue to use it in this article. Genetically
Brettanomyces shows a marked degree of diversity, which will
no doubt keep the taxonomists busy. Researchers looking at B.
bruxellensis have found large variation in chromosome size and
number4 and that it has a core diploid genome however
triploid strains are common5.

Vegetative Brettanomyces cells

Spore forming D.bruxellensis cells

Non-Saccharomyces yeast

As a non-Saccharomyces yeast its metabolism
differs from that of normal brewing yeast strains. It is able to use
a wider range nitrogen source, which gives it an advantage over
Saccharomyces spp. in nutritionally depleted environments such
as beer4. This has also been used as the basis for
selective media such as lysine agar6.

Brettanomyces will ferment glucose faster
in the presence of oxygen than it will anaerobically. This was named
the Custers effect after the researcher that discovered this in
19407. It will also produce acetic acid at the same time –
one of the reasons that Brettanomyces is often associated with
sour beer. However in an anaerobic environment this does not occur so
it is perfectly possible to make beers using Brettanomyces that
are not sour. As many beers fermented using Brettanomyces are
aged in wooden barrels or vats, it is worth noting that the larger
the vessel, the less oxygen ingress there is over time per unit
volume of beer8, which perhaps explains the fondness the
old porter brewers had for giant vats.

Carbon source

One of Brettanomyces best known
characteristics is its ability to attenuate beer further than normal
brewing yeast by utilising dextrins that they cannot ferment. Andrews
and Gilliland of the Guinness laboratory described how a secondary
attenuation limit was determined after fermentation with
Saccharomyces by using a culture of Brettanomyces9.
It can produce both an intra- and extra-cellular α-glucosidase, both
of which can potentially hydrolyse dextrins with greater than 9-12 glucose
units, producing glucose and the next lower dextrin10. In
a research project carried out at Heriot-Watt University, Chad
Yakobson found that glucose levels could even increase during
fermentation as this dextrin breakdown occurs11.

Sugar utilisation is highly variable within the
genus9,11. The slow growth of Brettanomyces means
that in mixed fermentations it is out competed by Saccharomyces
and cannot take full advantage of the wort glucose and maltose.
In pure culture Brettanomyces fermentations it is found that
most strains can however utilise both these sugars, though this does
to some extent repress dextrin utilisation and limit
super-attenuation12. Some strains produce a β-glucosidase
which allows them to ferment the wood sugar cellobiose. β-glucosidase
is also involved in the bio-transformation of hop compounds leading
to the release of glycosidically-bound flavour active volatile
compounds13.

Esters and esterases

Researchers looking into the complex world of
lambic fermentations have found that all the Brettanomyces
isolates they examined showed esterase activity not found in
Saccharomyces. The esterases found in Brettanomyces do
not only break down esters, they also have ester-synthesising
activity14. In lambic beers after Brettanomyces growth,
high levels of ethyl acetate (fruity, solvent flavour) and ethyl
lactate (fruity, creamy) were found, but very low levels of iso-amyl
acetate (banana). A similar situation is found in beers that have a
secondary Brettanomyces fermentation after primary
fermentation with Saccharomyces. The ester profile of the beer
is changed, something that will not happen if Saccharomyces is
used for conditioning. Other esters, such as ethyl caproate
(pineapple) and ethyl caprylate (fruity, winey, waxy) are also
associated with the flavour of lambic beers and are likely to be
produced by Brettanomyces15.

Phenolic compounds

Some of the characteristic flavour compounds
produced by Brettanomyces are phenols16. Flavour
active compounds produced include 4-vinylguiacol (clove flavour),
4-vinylphenol (barnyard, medicinal, plastic) and 4-vinylcatechol
(plastic, bitter, smoky). Unlike Saccharomyces, Brettanomyces has
the enzyme vinylphenol reductase which will reduce these compounds to
their corresponding ethyl derivatives3 :4-ethylguiacol (spicy, clove), 4-ethylphenol (medicinal) and
4-ethylcatechol (medicinal, barnyard). In beer the concentration of
4-ethylphenol is lower than 4-ethylguaicol, though in wine the
situation is reversed17.

Other flavour compounds

Mousy off-flavours can be caused by Brettanomyces
due to production of ETHP (2-ethyltetrahydropyridine) and ATHP
(2-acetlytetrahydropyridine). The amount produced is strain specific,
though the presence of oxygen stimulates their production4.
During anaerobic growth, Brettanomyces will produce a number
of fatty acids, some of which have cheesy or goaty flavours such as
isovaleric acid, caproic acid and caprylic acid3,11,15.

As can be seen not all the flavours produced by
Brettanomyces are desirable. A number of factors influence
which flavours are produced, these include the strain, pitching rate,
wort composition and fermentation conditions11,12.
Flavours can also change over time, as for example fatty acids are
esterified. When Brettanomyces beers are matured for a long
time they will require regular sampling to determine if the desired
flavour profile has developed. Some breweries will then use
pasteurisation when packaging to prevent further flavour changes12.

Fermentation

Brettanomyces can be used to carry out
fermentations in a number of different ways. When using B.
clausenii (WLP645) for secondary fermentations in a way similar
to that first described by Claussen, I found that the gravity of the
beer would drop by another two degrees Sacch (half a degree Plato)
and the fermentation would be complete after two months at room
temperature. As many brewers will be aware, a very small amount of
Brettanomyces can have a large effect and the recommended
pitching rate for secondary fermentations ranges from 100 to
2,000,000 cells per ml!12 I used approximately 500,000
cells per ml.

Pure Brettanomyces fermentations are slow
to start due to its inability to produce glycerol4 but
will still be completed within weeks with most strains giving an
apparent attenuation of 80-90%. The lack of glycerol production can
also make pure Brettanomyces beers taste thinner than normal.
As the Brettanomyces has easier access to nutrients and is
less stressed when no competing organisms are present, the flavour
development has been reported as more muted than that found when it
is used for secondary fermentations – though in my experience this
is not always the case. Pitching rates similar to that of
Saccharomyces are recommended for primary fermentation, and
some brewers oxygenate as normal whereas others to restrict
oxygenation to stress the yeast and increase production of flavour
compounds. Some brewers ferment at around 20°C, though others will
allow it to rise as high as 27-28°C to promote ester formation12.

In the “spontaneous” fermentations of lambic
and similar beers it can be eight months before the Brettanomyces
starts to out-compete the Saccharomyces, but once
established, it can continue to grow for years18. Growing
in mixed cultures with lactic acid bacteria leads to increased
attenuation9,19, as does adding lactic acid to the wort11.

Despite its long history in brewing, the full
potential of making beer with Brettanomyces is only now being
found and there is still much to learn about this interesting
organism.

Claussen, NH. (1904). On a Method for the
Application of Hansen's Pure Yeast System in the Manufacturing of
Well-Conditioned English Stock Beers. Journal of the Institute of
Brewing, Vol 10, Issue 4, 308-311.

Van Oevelen, D. et al. (1977).
Microbiological aspects of spontaneous wort fermentation in the
production of lambic and gueuze. Journal of the Institute of
Brewing, Vol 83, issue 6, 356-360.

Martens, H. et al. (1997).
Microbiological aspects of a mixed yeast-bacterial fermentation in
the production of special Belgian acid ale. Journal of the Institute
of Brewing, Vol 103, issue 2, 85-91.

This article apperared in the December 2016 issue of Brewer and Distiller International magazine. It was based on at talk I gave at the Carnivale Brettanomyces. Thanks to my colleagues Chris Rice and Chris Raleigh for the pictures of the cells, the picture of the D.bruxellensis is the first one I've seen of this bug when it's formed spores.

3 comments:

Interpreting it from a homebrewer's point of view, if I wanted to make something like a stock ale or a historic porter and age it with Brettanomyces then a) I should be fine using a glass carboy for the ageing and not worrying about trying to let oxygen in but b) the possibility that it'll end up tasting like old socks is a risk that I'll have to take. Is that about right?